人脐带间充质干细胞向视网膜色素上皮细胞分化的研究

发布时间：2018-05-12 19:35

本文选题：人脐带间充质干细胞 + 视网膜色素上皮细胞　；参考：《河北医科大学》2014年硕士论文

【摘要】：视网膜色素上皮(Retinalpigmentepithelium,RPE)，位于视网膜的最外层，为单层立方或矮柱状细胞。色素上皮来源于胚胎时期的神经外胚层，为光感受器细胞提供必需的营养并起到支持和保护的作用，同时为维持视网膜和感光细胞的正常生理功能提供多种营养因子。视网膜变性等损伤性疾病将导致视觉功能的损伤，尤其是视细胞的丢失将导致视觉功能的永久丧失。再生医学的发展使移植恢复视觉功能成为希望。近年来，学者将研究重点放在了视网膜细胞的移植上，特别是视细胞和视网膜色素上皮细胞的移植。各国学者先后应用胚胎干细胞（embryonic stem cells，ES)、诱导性多能干细胞（induced pluripotent stem cells，iPS)和成体干细胞中的脂肪来源干细胞诱导分化形成视网膜色素上皮样细胞，由于在培养过程中应用到了动物来源的血清、饲养层细胞、重组蛋白和营养因子，容易引起免疫反应，因此这些细胞的临床应用受到限制。在前期的研究中，我们在将ES诱导分化为视细胞的过程中外源性的添加了小分子化合物，没有使用动物来源的血清、生长因子和胚胎成纤维细胞饲养层，将此种方法诱导形成的视细胞进行移植实验，未发现有移植排斥反应，，并且细胞与宿主细胞的融合较好。这种诱导方法使视细胞移植的临床应用成为可能。人脐带Wharton胶来源的间充质干细胞（human Wharton’sjelly-derived mesenchymal stem cells，HWJ-MSCs）又称为脐带间充质干细胞(human umbilical cord mesenchymal stem cells，huc-MSCs)，多项研究显示，该细胞增殖和诱导分化能力很强；来源广泛且采集方便；不受道德伦理制约，是自体细胞移植和异体细胞移植治疗的理想种子细胞。与同样属于成体干细胞范畴的骨髓间充质干细胞（bone marrow mesenchymal stemcells，BM-MSC）相比，脐带间充质干细胞在取材上不受疾病、年龄等限制。脐带间充质干细胞在细胞移植的临床应用中具有更大优势。因此，本研究通过实验研究旨在探索相对成熟、稳定的体外分离培养、诱导脐带间充质干细胞向视网膜色素上皮样细胞分化的方法，为其最终应用于临床治疗提供实验依据。目的：在直接共培养体系中共培养SD大鼠睾丸支持细胞和人脐带间充质干细胞，探讨huc-MSCs向视网膜色素上皮细胞诱导分化的可行性，并对诱导分化的视网膜色素上皮细胞进行生物学特性鉴定，为后续的视细胞分化研究提供技术方法和实验基础。方法： 1已鉴定huc-MSCs的传代培养：常规培养经过鉴定的huc-MSCs。倒置显微镜观察细胞形态，选取第三代细胞进行共培养。 2SD乳鼠睾丸支持细胞（sertoli cells，SCs）的原代培养：选取19~21天龄的SD大鼠分离睾丸,低渗处理培养细胞进行纯化；倒置相差显微镜下观察纯化过程中细胞形态变化；吉姆萨染色、吖啶橙荧光染色、Feulgen染色鉴定SCs。 3huc-MSCs与SCs直接共培养：单层培养SCs至融合率80%；接种huc-MSCs并加入视黄酸进行共培养，形态学观察细胞生长情况；免疫荧光检测培养细胞中Pax6蛋白的表达；RT-PCR检测培养细胞中酪氨酸酶相关蛋白(tyrosinase-related protein2,TRP-2)，视网膜色素上皮细胞蛋白(Retinal pigment epithelium-specific65kDa protein，RPE65), Mer受体酪氨酸激酶（Proto-oncogene tyrosine-protein kinase MER, MERTK）和细胞视黄醛结合蛋白(cellular retinaldehyde-binding protein,CRALBP)的mRNA的表达。结果： 1第三代huc-MSCs呈长梭形，形态均一，生长状态良好。 2原代培养的SCs于第二天开始贴壁，培养七天后SCs纯度达95%以上；细胞形态结构特征与用伊红染色、吖啶橙荧光染色、Feulgen染色鉴定为SCs的形态结构特征一致。 3在huc-MSCs与SCs直接共培养体系中，培养一周后镜下观察细胞呈棕褐色，培养二周后细胞密度显著增高;免疫荧光法检测到细胞中有Pax6蛋白的表达；RT-PCR检测细胞内有Trp2、RPE65、Mertk和CRALBP的mRNA的表达。结论：huc-MSCs以睾丸支持细胞作为共培养细胞，加入视黄酸可以诱导分化为视网膜色素上皮细胞，方法简便可行。
[Abstract]:Retinal pigment epithelium ( RPE ) , which is located at the outermost layer of retina , is a single - layer cubic or low - columnar cell . The pigment epithelium is derived from neuroectoderm in embryonic period . It can provide the necessary nutrients for photoreceptor cells and play a role of support and protection . In recent years , we have focused on the transplantation of retinal cells . In recent years , scholars have focused on the transplantation of retinal cells , such as embryonic stem cells ( ES ) , induced pluripotent stem cells ( ES ) and embryonic fibroblast feeder layers .

Human umbilical cord - derived mesenchymal stem cells ( HWJ - MSCs ) are also known as human umbilical cord mesenchymal stem cells ( huc - MSCs ) .
wide sources and convenient acquisition ;
Compared with bone marrow mesenchymal stem cells ( BM - MSCs ) , umbilical cord mesenchymal stem cells are not limited by disease , age and so on .

Therefore , the aim of this study was to explore the method of differentiation of mesenchymal stem cells into retinal pigment epithelium - like cells in vitro and to provide experimental basis for clinical treatment .

Objective : To investigate the feasibility of cultured human umbilical cord mesenchymal stem cells ( MSCs ) and human umbilical cord mesenchymal stem cells ( MSCs ) in direct co - culture system , and to investigate the feasibility of huc - MSCs in inducing differentiation of retinal pigment epithelial cells , and to provide a technical and experimental basis for the subsequent study of the differentiation of retinal pigment epithelial cells .

Method :

1 . The culture of huc - MSCs has been identified : conventionally cultured huc - MSCs were cultured . The morphology of cells was observed by reversed microscope and the third generation cells were selected for co - culture .

The primary culture of the testis - supporting cells ( SCs ) of 2SD rats was carried out : the cells were isolated from 19 to 21 days old SD rats , and the cells were purified by hypotonic treatment .
The morphological changes of cells were observed under reversed phase contrast microscope .
The SCs were identified by fluorescence staining of acridine orange and Feulgen staining .

3huc - MSCs were co - cultured with SCs : single - layer cultured SCs to fusion rate of 80 % ;
huc - MSCs were inoculated and retinoic acid was added for co - culture and morphological observation of cell growth ;
Immunofluorescence was used to detect the expression of pa6 protein in cultured cells .
RT - PCR was used to detect the mRNA expression of tyrosinase - related protein ( RPE65 ) , retinal pigment epithelium - specific 65 kDa protein ( RPE65 ) , Mer receptor tyrosine kinase ( RPE65 ) , Mer receptor tyrosine kinase ( MERTK ) and cellular retinal aldehyde - binding protein ( CRAB ) in cultured cells .

Results :

The third generation of huc - MSCs had long shuttle shape , uniform morphology and good growth state .

2 primary cultured SCs began to adherent on the second day , and the purity of SCs reached over 95 % after seven days .
The morphological and structural features of the cells were stained with eosin , acridine orange fluorescence staining and Feulgen staining to identify the morphological characteristics of SCs .

3 In the direct co - culture system of huc - MSCs and SCs , the observed cells were brown - brown under the mirror of one week , and the density of cells increased significantly after two weeks .
The expression of Trp2 , RPE65 , Mertk , and CRACKI mRNA in the cells was detected by RT - PCR .

Conclusion : huc - MSCs can induce differentiation into retinal pigment epithelial cells by using testicular support cells as co - cultured cells , and the method is simple and feasible .

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R774.1

【参考文献】